Seroprevalence, molecular characterization, biotyping, and associated risk factors of bovine viral diarrhea in dairy cattle in Bangladesh.

Background and Aim:Bovine viral diarrhea (BVD) virus (BVDV) is an important viral pathogen of cattle that can infect diverse artiodactyl species. The clinical manifestations caused by BVDV in heterologous hosts, as they do in cattle, vary, although respiratory and reproductive failures are commonly reported. BVDV infections commonly result in reproductive failure in goats, with abortion being the primary clinical sign. In central Java, Indonesia, BVDV infection has been reported in two clinically healthy local goat species, and the testing indicated infection by BVDV Type 1. However, the genetic diversity of viruses has not been described in healthy or ill goats. The objectives of the present study were as follows: (1) To investigate the genetic variation of BVDV isolated from Sapera dairy goats with naturally occurring reproductive disorders in Yogyakarta, Indonesia, using the 5’ untranslated region (5’ UTR) and (2) to study the possible correlation between reproductive disorders and the presence of BVDV in the flock.Materials and Methods:Blood samples were collected in October 2021 from 39 goats that had been individually reported to have reproductive disorders. The serum samples were subjected to molecular detection and genetic characterization of BVDV based on the 5’ UTR of the viral genome, followed by sequencing and phylogenetic analyses. Viral isolation was performed on BVDV-positive samples to analyze the viral biotypes.Results:BVDV infection was detected in five out of 39 female goats. The clinical status of the BVDV-infected goats was abortion (n=2), metritis (n=1), and repeated breeding (n=2). All antigen-positive samples were confirmed as BVDV type 1a (BVDV-1a) and noncytopathic (NCP)-BVDV biotype.Conclusion:The BVDV-1a and NCP biotypes are the main subtypes and biotypes present in Sapera dairy goats exhibiting reproductive failure. This result is consistent with previous results in dairy cattle in Yogyakarta. The reported results can facilitate the design of methods for the prevention and control of BVD circulating in Indonesia.

AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross-contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA.

Serological studies have characterized the presence of the bovine viral diarrhea virus (BVDV) infection in water buffalo herds worldwide. However, the molecular characterization of BVDV strains circulating in this animal species is uncommon. The aim of this study was to identify young water buffalo with acute infection and characterize the subgenotype of the infecting wild-type BVDV strain. Two dairy water buffalo herds from Northeastern Brazil were selected based on the results of virus neutralization test which showed high titers of anti-BVDV antibodies. To identify viremic animals, the BVDV RNA was assessed by RT-PCR assay in serum samples from 44 asymptomatic young water buffalos, where 31 serum samples from herd A and 13 from herd B. Amplicons with 288bp of BVDV 5'UTR region were obtained in 7 (15.9%) serum samples (herd A, n = 5; herd B, n = 2). One good-quality amplicon from each herd was selected for nucleotide sequencing. The phylogenetic analysis demonstrated that the two BVDV wild-type strains clustered with BVDV strains of the subgenotype 1b. This study identified for the first time the active infection by BVDV subgenotype 1b in two dairy water buffalo herds from Brazil. These results highlight the importance of that, as well as in cattle herds, also in water buffalo herds prophylaxis measures to control BVDV infection should be intensified, mainly because these species clearly coexist in buffalo farms within Brazil.

Fifty-three cattle of unknown serologic status that were not persistently infected (PI) with bovine viral diarrhea virus (BVDV) were commingled with 10 cattle that were PI with different strains of BVDV, and were monitored for an extended commingle period using a reverse-transcription real-time PCR (RT-rtPCR) BVDV assay on various sample types. Transient infections with BVDV were also assessed by virus isolation, virus neutralization (VN) assays, and direct buffy coat 5'-UTR sequencing. Infections were demonstrated in all cattle by RT-rtPCR; however, the detection rate was dependent on the type of sample. Buffy coat samples demonstrated a significantly greater number of positive results ( p ≤ 0.05) than either serum or nasal swab samples. Presence of elevated BVDV VN titers at the onset inversely correlated with the number of test days positive that an individual would be identified by RT-rtPCR from buffy coat samples, and directly correlated with the average Ct values accumulated over all RT-rtPCR test days from buffy coat samples. Both single and mixed genotype/subgenotype/strain infections were detected in individual cattle by direct sample 5'-UTR sequencing. A BVDV-2a strain from a PI animal was found to be the predominant strain infecting 64% of all non-PI cattle; BVDV-1b strains originating from 3 PI cattle were never detected in non-PI cattle. Although direct sample 5'-UTR sequencing was capable of demonstrating mixed BVDV infections, identifying all strains suspected was not always efficient or possible.

Cattle immunotolerant to and persistently infected (PI) with bovine viral diarrhea (BVD) virus (BVDV) constitute the mechanism by which BVDV persists in and spreads among cattle herds. Detection and elimination of PI cattle are necessary for control of BVD. Serum is an excellent specimen for BVD PI testing because of high survivability of BVDV in serum and ease of collection, storage, and handling. Currently, microtiter virus isolation (VI) employing serum and sandwich ELISAs (S-ELISA) on tissues or leukocytes are used for BVDV PI screening. This paper evaluates a new S-ELISA kit that uses serum as the diagnostic sample. Cattle sera (n = 408) were tested using VI and the S-ELISA. The VI detected 172 BVDV-positive sera. Of these, 18 were confirmed PI cattle. The S-ELISA was positive on all PI samples. Considering only the PI animals, and using VI as the gold standard, the relative sensitivity of S-ELISA was 100%. The overall relative sensitivity was 93.6% and the agreement quotient (kappa) was 0.94. The relative specificity of the kit, based on 236 VI-negative sera, was 100%. These data indicate that the new kit is very adequate for detection of BVDV PI cattle.

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of the reproductive systems that have a significant socio economic impact on the dairy industry. A cross-sectional study was conducted on 17 randomly selected farms out of 133 registered dairy farms in Holeta, a town in the Walmera district of Ethiopia. The study aimed to detect BVDV antigen-positive animals and investigate the seroprevalence of BVDV infection. A total of 337 serum samples were collected from selected farms with no history of BVDV vaccination. The indirect enzyme-linked immunosorbent assay (I-ELISA) test was the screening test used to detect antibodies against BVDV. In contrast, antigencapture ELISA was used for the detection of BVDV antigens in seronegative animals. A logistic regression model was used to assess the association between potential predictors and BVDV exposure. The overall animal level seroprevalence was 15.4%, and 64.7% of the herds had at least one seropositive animal. From 285 seronegative animals, one animal (0.4%) was found positive for BVDV antigen. The same animal was found positive in a double check 21 days later. In this study, cows with a history of abortion (OR = 6.3; 95% CI: 1.61 -13.1), history of repeated breeding (OR = 7; 95% CI: 2.5 - 14.3), animals managed intensively (OR = 4.6; 95% CI: 1.6 - 13.0) and multiparous cows (OR = 3.6; 95% CI: 1.5 - 8.9) had higher proportion of seroreactors in their respective comparison category (p<0.05). Besides, cows with a history of congenital defective calve birth (OR = 15.2; 95% CI: 3.2 - 73.6), adult age groups (OR = 2.9; 95% CI: 1.0-7.9), and cows bred both artificially and natural mating (OR = 4.6; 95% CI: 1.7 - 12.6) were statistically associated with BVDV seropositivity (p<0.05). In conclusion, this report demonstrated the presence of persistently infected (PI) dairy cattle in Ethiopia. Thus, the findings warrant the need for immediate control intervention that involves both screening and culling of PI animals and vaccination.

Outbreak of persistently infected heifer calves with bovine viral diarrhea virus subgenotypes 1b and 1d in a BVDV-vaccinated open dairy herd

Background: Bovine viral diarrhea virus (BVDV) is one of the main agents that cause economical losses in cattle worldwide. Congenitally infected calves that are born persistently infected (PI) to BVDV are the main sources of infection to susceptible cattle. Direct contact is the most important form of transmission, but indirect contact can also spread BVDV, not only inside herds, but also between them. Transmission of BVDV by haematophagous insects has been proven experimentally, but the role of ticks in the transmission of BVDV has never been investigated. Ticks can heavily infest cattle raised in tropical areas and Rhipicephalus (Boophilus) microplus is the most important among them. The present experiment was carried out to investigate the role of R. microplus ticks in the transmission of BVDV, experimentally infecting PI calf with ticks. Material, Methods and Results: Three calves were used in the experiment: one PI calf was identified from a natural outbreak; a second animal was infested with the progeny of a tick fed on the PI calf and the third was kept as a negative control, infested with negative ticks. Viral RNA investigation was performed by reverse transcription followed by polymerase chain reaction (RT-PCR) from the sera of the calves and from ticks (adult females, eggs and larvae that were the progeny of the experimentally contaminated adult females and from the control animal). BVDV RNA was detected in tick adult females fed on the PI calf, but not in the control animal. Experimental infestation of a second cattle with larvae derived from adult females infected with BVDV was not able to produce infection. These data suggest that the virus is able to pass to ticks during feeding on the infected PI animal, but that there is no transmission by transovarial route, as viral RNA was not detected in eggs and larvae from adult females infected with BVDV. Discussion: Bovine tick is the most important ectoparasite in domestic animals in tropical and subtropical areas. However, its role in transmission of viral agents, particularly BVDV, has not been previously studied. The results of our experiment suggested that adult females of R. microplus were not able to transmit the infection to susceptible cattle. However, a macerate of a pool of tick females fed on the PI calf was positive to BVDV. A further validation using a larger number of infested bovines would help to confirm this new finding. R. microplus ticks are monoxenic, but it must be considered that the males, different from females, make a non-continual process of blood sucking and may move between bovines to reproduce. Additionally, in conditions of close contact between animals, besides tick males, larvae may change hosts in the early stages of development. These facts do not permit to exclude the risk of direct spread of viral infection in the herd by a same specimen of R. microplus. On the other hand, the presence of virus inside females represents an environmental reservoir of BVDV, which may infect through epithelial abrasions if ingested. These considerations may be reinforced by the fact that in field conditions it has been observed that high animal density favors the fast spread of BVDV in cattle herds. Collectively, these evidences suggest that ticks would represent an additional factor to be allowed for in BVDV transmission. As a conclusion, the present study demonstrates that R. microplus can be contaminated with BVDV during blood feeding, strengthening the idea that haematophagous vectors can be involved in the spread of the disease. In spite of the fact that BVDV was not transmited by the progeny of the ticks, it is not possible to discard such form of transmission under natural conditions.

A systematic review and meta-analysis of the epidemiology of bovine viral diarrhea virus (BVDV) infection in dairy cattle in China

Bovine Viral Diarrhea Virus (BVDV) is a single-stranded RNA virus in the Pestivirus genus within the Flaviviridae family. BVDV infections are seen in all ages and breeds of cattle worldwide and have significant economic impact due to productive and reproductive losses (Houe 2003). Two antigenically distinct genotypes of BVDV exist, type 1 and 2 (Ridpath et al. 1994). BVDV of both genotypes may occur as cytopathic (cp) or noncytopathic (ncp) biotypes, classified according to whether or not they produce visible changes in cell culture. Data indicate that cp biotypes of BVDV can actually be created through internal deletion of RNA of ncp biotypes, or through RNA recombination between ncp biotypes (Howard et al. 1992). Of the two BVDV biotypes, infection of a fetus by ncp BVDV can result in persistently infected (PI) calf that sheds the virus throughout its life without developing clinical signs of infection. PI animals are the major disseminators of BVDV in the cattle population and have been the cause of severe acute outbreaks (Carman et al. 1998). However cp BVDV is associated predominantly with animals that develop mucosal disease (MD), which can be acute, resulting in death within a few days of onset, or chronic, persisting for weeks or months before the afflicted animal dies (Houe 1999). The interaction of BVDV with its host has several unique features, most notably the capacity to infect its host either transiently or persistently (Liebler-Tenorio et al. 2002; Bendfeldt S 2007). Initially the virus binds to CD46, a complement receptor expressed on lymphoid cells, monocytes, macrophages and dendritic cells and serving as a “magnet” for several viral and bacterial pathogens (Cattaneo 2004). Upon entry, the virus replicates and spreads in the lymphatic system, impairing the immunity of the infected animal, particularly antigen presenting cells (APC) function and production of interferons (IFN). Cytopathic BVDV biotype but not ncp biotype (Schweizer & Peterhans 2001) is implicated in the induction of apoptosis (Zhang et al. 1996; Schweizer & Peterhans 1999; Grummer et al. 2002; Jordan et al.

The identification and elimination of persistently infected (PI) cattle are the most effective measures for controlling bovine pestiviruses, including bovine viral diarrhea virus (BVDV) and the emerging HoBi-like viruses. Here, colostrum-deprived calves persistently infected with HoBi-like pestivirus (HoBi-like PI calves) were generated and sampled (serum, buffy coat, and ear notches) on the day of birth (DOB) and weekly for 5 consecutive weeks. The samples were subjected to diagnostic tests for BVDV--two reverse transcriptase PCR (RT-PCR) assays, two commercial real-time RT quantitative PCR (RT-qPCR), two antigen capture enzyme-linked immunosorbent assays (ACE), and immunohistochemistry (IHC)--and to HoBi-like virus-specific RT-PCR and RT-qPCR assays. The rate of false negatives varied among the calves. The HoBi-like virus-specific RT-PCR detected HoBi-like virus in 83%, 75%, and 87% of the serum, buffy coat, and ear notch samples, respectively, while the HoBi-like RT-qPCR detected the virus in 83%, 96%, and 62%, respectively. In comparison, the BVDV RT-PCR test had a higher rate of false negatives in all tissue types, especially for the ear notch samples (missing detection in at least 68% of the samples). The commercial BVDV RT-qPCRs and IHC detected 100% of the ear notch samples as positive. While ACE based on the BVDV glycoprotein E(rns) detected infection in at least 87% of ear notches, no infections were detected using NS3-based ACE. The BVDV RT-qPCR, ACE, and IHC yielded higher levels of detection than the HoBi-like virus-specific assays, although the lack of differentiation between BVDV and HoBi-like viruses would make these tests of limited use for the control and/or surveillance of persistent HoBi-like virus infection. An improvement in HoBi-like virus tests is required before a reliable HoBi-like PI surveillance program can be designed.

To compare the efficacy of modified-live virus (MLV) vaccines containing either type 1 bovine viral diarrhea virus (BVDV) or types 1 and 2 BVDV in protecting heifers and their offspring against infection associated with heterologous noncytopathic type 2 BVDV challenge during gestation. Randomized controlled study. 160 heifers and their offspring. After inoculation with a placebo vaccine, 1 or 2 doses of an MLV vaccine containing type 1 BVDV, or 1 dose of an MLV vaccine containing both types 1 and 2 BVDV, heifers were bred naturally and challenge exposed with a type 2 BVDV field isolate between 62 and 104 days of gestation. Pregnancies were monitored; after parturition, virus isolation and immunohistochemical analyses of ear-notch specimens were used to determine whether calves were persistently infected. Blood samples were collected at intervals from heifers for serologic evaluation and virus isolation. Persistent infection was detected in 18 of 19 calves from heifers in the control group and in 6 of 18 calves and 7 of 19 calves from heifers that received 1 or 2 doses of the type 1 BVDV vaccine, respectively. None of the 18 calves from heifers that received the type 1-type 2 BVDV vaccine were persistently infected. Results suggest that the incidence of persistent BVDV infection among offspring from dams inoculated with 1 dose of the MLV vaccine containing types 1 and 2 BVDV was decreased, compared with 1 or 2 doses of the MLV vaccine containing only type 1 BVDV.

In this study, blood serum and leukocyte samples were collected from 400 Holstein heifers, all of which appeared to be healthy. Antibodies (Ab) against bovine viral diarrhea virus (BVDV) were detected in 57 serum samples, and BVDV antigen (Ag) was detected in 38 leukocyte samples. There were statistically important differences between the average first insemination ages (FIT) of the BVDV (Ag-/Ab+) heifers (p<0.0001) (pregnant p<0.05, nonpregnant p<0.0001) and BVDV (Ag-/Ab-) heifers. The average conception rates (CR) of BVDV (Ag-/Ab+) heifers and BVDV (Ag-/Ab-) heifers were not significant statistically. There were statistically important differences in average FIT between persistent infected (PI) BVDV (Ag+/Ab-) heifers (p<0.0001; PI pregnant p<0.05, PI nonpregnant p<0.0001) and BVDV (Ag-/Ab-) heifers. No significant differences in average CR between PI BVDV (Ag+/Ab-) heifers and BVDV (Ag-/Ab-) heifers were found. The differences in average FIT between BVDV (Ag+/Ab+; p<0.0001; nonpregnant p<0.0001) and BVDV (Ag-/Ab-) heifers were important statistically. Although there were no BVDV (Ag+/Ab+) pregnant heifers, the differences in average CR between BVDV (Ag+/Ab+) pregnant heifers and BVDV (Ag-/Ab-) heifers were found to be statistically important (p<0.0001). We conclude that fertility is affected in heifers with BVDV (Ag-/Ab+, Ag+/Ab- and Ag+/Ab+).

Induction of interferon-gamma and downstream pathways during establishment of fetal persistent infection with bovine viral diarrhea virus

Background: Infections are caused by Bovine Viral Diarrhea Virus and still continue to be a worldwide plague in cattle industry. It is responsible for sudden death syndromes in adult cattle with high mortality rates, abortions, acute gastrointestinal and respiratory diseases. The BVDV infection occurs in early pregnancy (40-142 days), in immunosuppressed females or cows results in 100% of persistently infected (PI) calves that are seronegative and asymptomatic at birth. Evidences suggests that BVDV contributes to BRD complex potentiating secondary infections caused by Mannheimia haemolytica e Pasteurella multocida due to its immunosuppressive action. However, the farmers have often associated the respiratory syndrome with other infectious agents. This paper reports the attendance of dairy calves manifesting clinical signs of bronchopneumonia, which led to the screening of the persistently infected animals to control of the BVDV infection in the herd.Materials, Methods &amp; Results: During the technical assistance, ten calves manifesting bronchopneumonia were selected to trans-tracheal lavage (TL) in order to identify possible infectious agents. Reverse transcription polymerase chain reaction (RT-PCR) detected the presence of BVDV in two heifers. Pasteurella multocida was the unique bacterial agent isolated from TL (5/10, 50%). These data motivated the technical team and producers to investigate the PI screening by direct enzyme-linked immunosorbent assay from biopsies of the ear edge. The screening of PI’s detected 29 positive within of 2,342 animals tested (1.23%). The re-test of positive was performed only in 24 animals due to the cull of five bovine with severe bronchopneumonia and diarrhea, confirming 18 persistently infected calves (18/24; 75%). Finally, in all PI’s live dams were tested. It was observed four positive adult animals. One grand dam was live and tested, but it had negative result for direct enzyme-linked immunosorbent test. The rate of PI’s considering the whole herd was 0.81% (22/3,700 animals).Discussion: The involvement of BVDV in the etiology of bronchopneumonia was confirmed by detection of the virus in trans-tracheal lavages in two calves by RT-PCR. The susceptibility for Pasteurella multocida infection could be promoted by BVDV prime-infection, considering that immunossupressive nature of BVDV is a critical factor in the interaction with others viruses and bacteria. At this time, we are aware about any report about the detection of BVDV in trans-tracheal lavages. These findings culminated with the screening of PI animals in the herd, detecting rates of 0.81%. The intensive vaccination and colostrum management of this farm could protected the herd against BVDV, however others facts facilitated the introduction of the virus in the herd. This research was conduced in a high-production dairy farm with around 3,700 animals raised in an open herd, in which some of cows with high genetic potential were transferred for embryo collection in the state of Paraná, Brazil; resulting in the addition of the calves to the herd by others routes. Moreover, the farm used for many years vaccine containing only BVDV-1, which may have favored the entry and spread of BVDV-2 or BVDV-3 in the herd. This research showed the presence of BVDV in trans-tracheal lavage of heifers with bronchopneumonia by RT-PCR. This fact points to the need of BRD control programs that include detection of PI animals.